Restoration of lysosomal acidification rescues autophagy and metabolic dysfunction in non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the world. High levels of free fatty acids in the liver impair hepatic lysosomal acidification and reduce autophagic flux. We investigate whether restoration of lysosomal function in NAFLD recovers autophagic flux, mitochondrial function, and insulin sensitivity. Here, we report the synthesis of novel biodegradable acid-activated acidifying nanoparticles (acNPs) as a lysosome targeting treatment to restore lysosomal acidity and autophagy. The acNPs, composed of fluorinated polyesters, remain inactive at plasma pH, and only become activated in lysosomes after endocytosis. Specifically, they degrade at pH of ~6 characteristic of dysfunctional lysosomes, to further acidify and enhance the function of lysosomes. In established in vivo high fat diet mouse models of NAFLD, re-acidification of lysosomes via acNP treatment restores autophagy and mitochondria function to lean, healthy levels. This restoration, concurrent with reversal of fasting hyperglycemia and hepatic steatosis, indicates the potential use of acNPs as a first-in-kind therapeutic for NAFLD.

(3) Mouse data are predominantly about improvements of insulin-glucose homeostasis, which are not substantiated by in vitro results. Dose acNP restore insulin signaling resposiveness of HepG2 cells?
Reviewer #2 (Remarks to the Author): NCOMMS-20-24293 Restoration of Lysosomal Acidification Rescues Autophagy and Metabolic Dysfunction in Nonalcoholic Fatty Liver Disease This study reports an approach to treat non-alcoholic fatty liver disease (NAFLD) using engineered polymeric nanoparticles that can be degraded in lysosomes and recover acidic condition restoring autophagy and metabolic dysfunction. The novelty of this work is the polymer design that allows selective degradation at dysfunctional lysosomal pH and effectively lowers the pH. The authors convincingly demonstrate the potential of this approach in reversing the effects of NAFLD by rescuing autophagic flux in vitro and in vivo. The treatment of NAFLD mice with acNPs led to significantly reduced steatosis, reversal of insulin resistance, as well as decrease in liver weight, and suggests that acNPs has potential as a treatment option for NAFLD. The manuscript could be strengthened with responses to these major comments: 1. For the general reader of Nature Communications, who might not be familiar with NAFLD and its mechanism, it would be helpful to present a schematic diagram that covers the major pathway addressed here, from lysosomal acidification restored by polymeric nanoparticles and its therapeutic outcomes. 2. The second paragraph of the introduction is almost identical to the introduction of a previous publication by the author with sections that are copied word-for-word (highlighted in manuscript). 3. Curiously, the authors do not show a correlation between polymer degradation, and rate of acidification, which would seem to be the most important structure/function relationship here. A study of nanoparticle degradation (e.g. DLS) and/or polymer degradation (e.g. GPC) should be included to show that pH change correlates with particle degradation. In particular, is the PESU control particle not degrading at all or is the pKa of succinic acid too high to cause any change? As the polymerization of PEFSU is not completely random according to NMR (with sections of homopolymerized succinic acid) maybe the particle/polymer is not completely degrading and only sections of homopolymerized TFSA on the surface are degrading while there are still sections of undegraded TFSA embedded in the nanoparticle core? 4. Why is this particular ratio of TFSA to SA used and not for example a pure homopolymer entirely composed of TFSA? If other ratios were tested for their pH response, it would be helpful to list those studies in the supplementary information. 5. It would be interesting to see how acNPs affect the pH of normally functioning acidic lysosomes. In particular, would this lead to an excessively low pH? 6. There are many animal models for NAFLD, and the phenotypes are mostly similar but definitely varied. The therapeutic effects of acidic nanoparticles can be also varied, depending on the animal model, nature of induction of disease, and extent of disease progression. Please address the rationale or discussion regarding the specific model used here. 7. The time chart describing animal experiment from disease induction and its treatment should be presented in one of the main figure, since there are groups with different doses and multiple doses. 8. Most nanoparticles-regardless of composition-accumulate in the liver after i.v. administration. However, their distribution within liver tissue are not uniform. In most cases, the majority of particles are taken up by Kupffer cells, not hepatocytes. Considering nanoparticle distribution and their poor diffusion in tissue, it could be unclear how nanoparticles affect lysosome and rescue autophagy. Distribution of nanoparticles using fluorescent microscope imaging should be supported with other types of analysis, such as flow cytometry data. 9. Previous work on nanoparticle-mediated lysosomal acidification should be discussed in more detail. Lysosomal acidification was attempted before in other publications (including by this group) using photoactivated nanoparticles and PLGA nanoparticles with similar studies being conducted. 10. The relationship between lysosome acidification and autophagosome fusion should be discussed. How does lysosome re-acidification rescue autophagosome fusion? If reduced autophagosome fusion is an independent effect of NAFLD how does lysosome acidification affect this?
Other minor comments: Introduction-Nanoparticles are not always monodisperse and around 100 nm, as suggested here. Results-What is the pKa of succinic acid, and what role does that play in the action of the particles? The method used to generate data in Fig 1C is not described in the Methods. Why don't PESU particles change the pH? Is this because the particles are not degrading (fast enough) or because the pKa of degradation product (succinic acid) is too high to have any effect? In Fig 5A, how long was treatment to achieve this reduction in liver weight? Some of the data in  Authors investigated a new biodegradable acid-activated acidic nanoparticles (acNPs) as a lysosome targeting strategy to manipulate lysosomal acidity and autophagy in hepatocytes and in experimental non-alcoholic fatty liver disease (NAFLD) mouse model. They were able to show that acNPs, composed of fluorinated polyesters, improved lysosomal acidity and rescued lysosomal function to some extent in cultured human hepatoma cells and in mouse livers. In vivo administration of acNP also can improve diet-induced insulin sensitivity and steatosis likely via increased autophagic flux and mitochondrial functions. While this tool has held a promise to treat lysosomal defects mediated diseases such as NAFLD, the study is largely descriptive in nature. The autophagic flux data and mitophagy were weak and not convincingly support the conclusions.
Specific comments: 1. Figure 2, in addition to the change of lysosome size, it seems that the number of lysosomes also altered. The authors claimed that the size changes could be due to increased turnover via autophagy. However, more data are needed to support this. Authors should quantify the number of lysosomes or more quantitative manner for lysosomal proteins. Lysosomal stress such as pH changes often leads to the activation of TFEB, a master regulator for lysosomal biogenesis gene transcription. Would altered lysosomal pH affect TFEB-mediated transcription program? 2. Figure 2E, authors should add a lysosomal inhibitor such as Bafliomycin A or leupeptin to confirm the autophagic flux changes of LC3-II. To better support the lipid changes, the total levels of triglyceride should be measured in Figure 2I. 3. Palmitate is toxic to HepG2 cells. Would improved lysosomal functions by acNP affect palmitate lipotoxicity? Ideally these experiments should be repeated in primary cultured hepatocytes as HepG2 cells are cancerous in nature. 4. Figure S3C, the figure labeled as LAMP-1 but in the text it was stated as LAMP-2? 5. More experimental details should be provided for Fig S4. 6. Figure 6, the restoration of autophagy function by using western blot for LC3-II and p62 could be troublesome as both protein could be regulated at the transcription level. Also decreased LC3-II could also be due to decreased formation of autophagosomes. Adding a lysosomal inhibitor such as leupeptin in these experiments will help to clarify these issues. In addition, no data provided to show improved lysosomal functions by acNP in mouse NAFLD models. 7. Inflammation is critical for the progression of NAFLD to NASH. Authors should provide data to show whether acNP can also affect liver non-parenchyma cells (such as macrophages) in addition to hepatocytes. It is highly likely a large amount of acNP would be taken up by macrophages/Kupffer cells in the liver. The function of these macrophage/Kupffer cells after acNP should be determined. 8. Figure 6, mitophagy data were very weak. First the control LFD group was missing, and it was unclear whether HFD would impair mitophagy in this model. The change of mitoTracker could be due to various factors such as mitochondrial membrane potential and may not be a good marker for mitophagy. Mitophagy refereed to more specific autophagic removal of damaged mitochondria. Did the authors observed lysosomes that contain mitochondria? If HFD impaired lysosomal pH and functions, one would observe more mitochondria in the lysosomes? And acNP treatment should lead to few mitochondria inside lysosomes. The changes of more mitochondrial proteins should also be included.

Reviewer #1 (Remarks to the Author):
The paper reports a nanoparticle that has a novel activity in lowering lysosomal pH and therefore improving autolysosomal homeostasis. When treated to HepG2 cells, this nanoparticle can lower lysosomal pH and increase lysosomal enzymatic activities. It also decreases accumulation of LC3-II and p62, as well as lipid droplets, in palmitate-induced lipotoxic condition. When treated in vivo, it localizes in liver, and improves glucose homeostasis and hepatosteatosis.
Although this is an interesting report, several key experiments need to be conducted to support the main claims.
We thank the reviewer for noting that our work is interesting, and we have performed the suggested additional experiments to substantiate the findings (see below).
(1) acNP effects on autophagic flux needs to be shown, in both HepG2 cells and in vivo mouse livers.
Currently, only the steady-state levels of LC3 and p62 were shown.
Response: Thank you for the comment and we have performed additional experiments at non steady-state conditions. We have determined the autophagic flux in HepG2 cells via comparing the effects of acNPs addition before and after adding either a lysosomal V-ATPase/acidification inhibitor, bafilomycin A1 (100 nM for 2 hours, Baf), or a lysosomal protease inhibitor, leupeptin (100 uM for 24 hours, Leu) ( Fig.   3E -G). Bafilomycin disruption of lysosomal acidification resulted in dysfunctional autophagic clearance of autophagosome, hence leading to an accumulation of autophagosomes (e.g., elevation of LC3-II and p62 levels). The addition of leupeptin also resulted in a slight accumulation of LC3-II and p62 levels compared to the acNPs treated condition, although at a lower level than with bafilomycin treatment.
These results suggest that the effect of acNPs in modulating autophagic flux is mediated mainly through affecting lysosomal acidification, and not due to modulating the lysosomal protease degradative activity.
We have henceforth also chosen bafilomycin as the standard control in the subsequent experiments.
The study of autophagic flux in mouse livers using lysosomal acidification inhibitors (e.g., chloroquine, bafilomycin) is limited by the toxicity of these treatments (e.g., cardiac, neurotoxicity and retinal toxicity and cell death) 1 .Primary human hepatocytes are considered the gold standard short-term human in vitro liver model because of their high functionality relative to the human organ in vivo 2 . Hence, we chose to use primary human hepatocytes and analyzed the turnover of mitochondria (e.g., mitophagy) to determine autophagic flux in vivo. Using primary human hepatocytes, we show that under palmitate conditions (recapitulate HFD condition in mice), mitochondrial content is increased while treatment with acNPs decreases it, indicating increased mitochondrial degradation (Fig. S7K). The maximal respiratory rate (MRR) of mitochondria decreases under palmitate condition, while the addition of acNPs restores the MRR (Fig. S7L). When we measured the oxygen consumption rate (OCR) of mitochondria with and without lysosomal acidification with bafilomycin or acNPs, the OCR of mitochondria decreases upon bafilomycin treatment, while acNPs treatment restores OCR (Fig. S7M). In sum, these data indicate that the effect of acNPs in restoring mitochondrial respiratory function is due to improving lysosomal acidification function and autophagic turnover of mitochondria.
(2) Whether acNP restores in vivo lysosomal homeostasis is not shown. It is unclear whether acNP corrects metabolism through restoring liver metabolism. To address this, hepatocyte lysosomal acidification and lysosomal enzyme activities should be measured in control, LD and HD mice.
Response: Currently, there are no tools to directly measure lysosomal acidification and lysosomal enzyme activities in vivo. Therefore, we acknowledge this is a limitation in the study. To pinpoint the effect of lysosomal acidification on lysosomal homeostasis in vivo, we have treated primary human hepatocytes in the presence of bafilomycin or palmitate, with or without acNPs, and measured the lysosomal activity using Magic red assay (fluorescence increases intensity as lysosomal cathepsin L activity increases) ( Fig.   S7N -O). Treatment with either Bafilomycin A1 or palmitate decreases Magic red intensity, indicating decreased lysosomal enzyme activity. Treatment with low dose (LD) or high dose (HD) acNPs increases the magic red intensity, and HD acNPs show a more significant increase. These results indicate that the increase in lysosomal enzyme activity is due to changes in lysosomal acidification upon acNPs treatment. This study reports an approach to treat non-alcoholic fatty liver disease (NAFLD) using engineered polymeric nanoparticles that can be degraded in lysosomes and recover acidic condition restoring autophagy and metabolic dysfunction. The novelty of this work is the polymer design that allows selective degradation at dysfunctional lysosomal pH and effectively lowers the pH. The authors convincingly demonstrate the potential of this approach in reversing the effects of NAFLD by rescuing autophagic flux in vitro and in vivo. The treatment of NAFLD mice with acNPs led to significantly reduced steatosis, reversal of insulin resistance, as well as decrease in liver weight, and suggests that acNPs has potential as a treatment option for NAFLD.
We thank the reviewer for noting the novelty of our work.
The manuscript could be strengthened with responses to these major comments: 1. For the general reader of Nature Communications, who might not be familiar with NAFLD and its mechanism, it would be helpful to present a schematic diagram that covers the major pathway addressed here, from lysosomal acidification restored by polymeric nanoparticles and its therapeutic outcomes.
Response: Thank you for the comment. A schematic which links lysosomal acidification to autophagy, mitochondrial function, lipid accumulation, and inflammation in the pathogenesis of NAFLD, as well as how NPs treatment affects these processes, has been included in Figure. 1 of the revised manuscript. 3. Curiously, the authors do not show a correlation between polymer degradation, and rate of acidification, which would seem to be the most important structure/function relationship here. A study of nanoparticle degradation (e.g. DLS) and/or polymer degradation (e.g. GPC) should be included to show that pH change correlates with particle degradation. In particular, is the PESU control particle not degrading at all or is the pKa of succinic acid too high to cause any change? As the polymerization of PEFSU is not completely random according to NMR (with sections of homopolymerized succinic acid) maybe the particle/polymer is not completely degrading and only sections of homopolymerized TFSA on the surface are degrading while there are still sections of undegraded TFSA embedded in the nanoparticle core?
Response: We have included a GPC study to analyze the molecular weight of the polymers after degradation in pH 6.0 buffer. We observed a correlation between polymer degradation and buffer acidification. The 25% PEFSU polymer degrades rapidly in the first 24 hours, followed by a much slower rate of degradation within the next 24 hours (Fig. 2E). We have also used scanning electron microscopy to monitor the size changes of the nanoparticles across 48 hours, where the nanoparticles size increases within the first 24 hours, potentially indicative of bulk degradation (Fig. S1H -J). This aligns with other studies describing polyester (e.g., PLGA) nanoparticle degradation with an initial particle swelling before a decrease in nanoparticle size 3  Response: We thank the reviewer for the suggestion. We have previously tried to make a pure homopolymer entirely composed of TFSA, however the polymerization was unsuccessful despite optimization using different reaction conditions and parameters. We have included a table of polymers that we have successfully synthesized with different ratios of TFSA and SA, as well as with different glycol linker groups (Fig. A). We have also determined the pH modulation capability of these polymers, and it was shown that 25% TFSA to 75% SA (25% PEFSU) had the most significant pH modulation property (Fig. B). We will discuss the studies briefly in the manuscript text, but we do not intend to include these data in the supplementary information, as these results will be discussed in greater detail in subsequent publications focused mainly on the polymer chemistry.
5. It would be interesting to see how acNPs affect the pH of normally functioning acidic lysosomes. In particular, would this lead to an excessively low pH?
Response: We have measured the lysosomal pH in HepG2 cells with acNPs treatment in BSA media and the pH does not change significantly with respect to the BSA control cells (Fig. S2A -B).
6. There are many animal models for NAFLD, and the phenotypes are mostly similar but definitely varied. The therapeutic effects of acidic nanoparticles can be also varied, depending on the animal model, nature of induction of disease, and extent of disease progression. Please address the rationale or discussion regarding the specific model used here.
Response: Mice and rats have been used most frequently in NAFLD modeling. The C57BL/6 strain in mice and Wistar and Sprague Dawley strains in rats are generally preferred because of their intrinsic preference to develop obesity and NAFLD 4,5 . Hence, we have used C57BL/6 strain mice as our high-fat diet (HFD) model. There have been multiple diets used to generate NAFLD phenotypes, including MCD diet, Choline-Deficient l-Amino Acid-defined Diet, Atherogenic Diet, Fructose and High-fat diet (HFD). We chose the HFD fed model with fat content of 60 kcal%, as it has been shown to bring about a phenotype similar to the human disease, characterized by obesity (after 10 weeks), insulin resistance (hyperinsulinemia after 10 weeks and glucose intolerance after 12 weeks) and hyperlipidemia (after 10 weeks) 6,7 . 16 weeks of HFD regimen was chosen as it recapitulates liver steatosis found in NAFLD, and evidence suggests that mice fed with HFD for 16 weeks had decreased mitochondrial respiration and bioenergetics compared with control mice 8 . We have included this additional information on animal model choice in the revised manuscript. Response: We studied the co-localization of Rhodamine labelled acNPs in HFD mice with Kupffer cells stained with alexa-fluor-647-anti-mouse-clec4f-antibody (BioLegend) upon tail vein injection. Through quantification of the immunofluorescence images, we observed that only 20% of the Rho-acNPs colocalized with MAC2 positive cells, while 80% of the Rho-acNPs resided in hepatocytes or the surrounding tissues. Hence, this indicates that the effect of acNPs on autophagic function as well as liver metabolic function is largely due to its action in hepatocytes. These data are included in Figure. S3C. 9. Previous work on nanoparticle-mediated lysosomal acidification should be discussed in more detail.
Lysosomal acidification was attempted before in other publications (including by this group) using photoactivated nanoparticles and PLGA nanoparticles with similar studies being conducted.
Response: We thank the reviewer for the suggestion. We have included the discussion of previous work on nanoparticle-mediated lysosomal acidification using the photoactivated nanoparticles and PLGA nanoparticles in the revised manuscript. While the photoactivated NPs show significant effect in restoring lysosomal acidity in pancreatic beta cells under lipotoxicity (type II diabetes model) 9 , the requirement of a UV-light trigger to activate the nanoparticles renders it inapplicable for in vivo applications. PLGA NP has been used to modulate lysosomal pH in a variety of disease models 10,11 . The acNP exhibit a 4 times more significant lysosomal pH reduction in HepG2 cells under palmitate treatment than PLGA NP at the same concentration (Fig. S8A). In addition, acNP causes a more significant reduction in lipid droplets accumulation in HepG2 cells, potentially due to a more significant decrease in lysosomal pH (Fig. S8B). 10. The relationship between lysosome acidification and autophagosome fusion should be discussed. How does lysosome re-acidification rescue autophagosome fusion? If reduced autophagosome fusion is an independent effect of NAFLD how does lysosome acidification affect this?
In NAFLD, increases in intracellular lipids (e.g., saturated fatty acids such as palmitic acid) have been shown to alter the intracellular membrane lipid composition of both autophagosomes and lysosomes.
Hence, this reduces the ability of autophagosomes to fuse with lysosomes and a subsequent decrease in autophagic flux 12 . Lysosomal pH has been shown to play a role in modulating autophagosome-lysosome fusion. In our previous work using photo-activated nanoparticles (paNPs) to re-acidify lysosomes in Other minor comments: Introduction-Nanoparticles are not always monodisperse and around 100 nm, as suggested here.
Response: We agree and changed the corresponding text.
Results-What is the pKa of succinic acid, and what role does that play in the action of the particles?
The method used to generate data in Fig 1C is not described in the Methods. Why don't PESU particles change the pH? Is this because the particles are not degrading (fast enough) or because the pKa of degradation product (succinic acid) is too high to have any effect?
Response: The pKas of succinic acid are 4.21 and 5.64. The polymer formed from pure SA has a high Tm as compared to 25% PEFSU, hence, within the time frame of our assay, the PESU polymer does not have significant degradation and does not generate significant changes in the lysosomal pH. We have included the method to generate data in Fig. 2C (previously Fig. 1C) in the materials and methods section.
In Fig 5A, how long was treatment to achieve this reduction in liver weight?
Response: The treatment was for three times (e.g., day 1, day 3 and day 5). We have also added this detail in the newly included timeline (Fig. 4A) for this experiment in the revised manuscript.
Some of the data in Fig 5 could be moved to Supplementary Data to improve readability.
Response: We have moved the liver weight/body weight % data for both HFD and LFD conditions in Fig.   5 to the supplementary data to improve readability.
In Fig 6, not all of the changes reached statistical significance, but this is not clear from the text.
Response: We have changed our text to describe these changes, as well as discussion of the results.

Reviewer #3 (Remarks to the Author):
Authors investigated a new biodegradable acid-activated acidic nanoparticles (acNPs) as a lysosome targeting strategy to manipulate lysosomal acidity and autophagy in hepatocytes and in experimental nonalcoholic fatty liver disease (NAFLD) mouse model. They were able to show that acNPs, composed of fluorinated polyesters, improved lysosomal acidity and rescued lysosomal function to some extent in cultured human hepatoma cells and in mouse livers. In vivo administration of acNP also can improve diet-induced insulin sensitivity and steatosis likely via increased autophagic flux and mitochondrial functions. While this tool has held a promise to treat lysosomal defects mediated diseases such as NAFLD, the study is largely descriptive in nature. The autophagic flux data and mitophagy were weak and not convincingly support the conclusions.
We thank the reviewer for the critical comments and we have performed additional experiments to support our findings.
Specific comments: 1. Figure 2, in addition to the change of lysosome size, it seems that the number of lysosomes also altered.
The authors claimed that the size changes could be due to increased turnover via autophagy. However, more data are needed to support this. Authors should quantify the number of lysosomes or more quantitative manner for lysosomal proteins. Lysosomal stress such as pH changes often leads to the activation of TFEB, a master regulator for lysosomal biogenesis gene transcription. Would altered lysosomal pH affect TFEB-mediated transcription program?
Response: We have quantified the number of lysosomes for each treatment condition. There is a decrease in the average number of lysosomes under palmitate treatment, however; the changes observed across treatment conditions are not statistically significant (see Figure. S2C). In a similar study done by our group studying the change in lysosomal acidification and lysosome number in pancreatic β cells under palmitate treatment, we showed that there is no change in the total lysosomal mass/number before and after palmitate treatment, as determined by LAMP-1 staining, but there is lysosomal pH increase after palmitate treatment, indicative that the change in lysosomal acidification does not affect lysosome number 13 .
2. Figure 2E, authors should add a lysosomal inhibitor such as Bafliomycin A or leupeptin to confirm the autophagic flux changes of LC3-II. To better support the lipid changes, the total levels of triglyceride should be measured in Figure 2I.
Response: Thank you for the comment. We have determined the autophagic flux in HepG2 cells via comparing the effects of acNPs addition before and after adding either a lysosomal V-ATPase/acidification inhibitor, bafilomycin A1 (100 nM for 2 hours, Baf), or a lysosomal protease inhibitor, leupeptin (100 uM for 24 hours, Leu) (Fig. 2E -G). Bafilomycin disruption of lysosomal acidification results in dysfunctional autophagic clearance of autophagosome, hence leading to an accumulation of autophagosomes (e.g., elevation of LC3-II and p62 levels). The addition of leupeptin also results in a slight accumulation of LC3-II and p62 levels compared to the acNPs treated condition, although at a lower level than with bafilomycin treatment. These results suggest that the effect of acNPs in modulating autophagic flux is mediated mainly through affecting lysosomal acidification, and not due to modulating the lysosomal protease degradative activity. We have also measured the total levels of triglyceride in HepG2 cells (Fig. S2E).
3. Palmitate is toxic to HepG2 cells. Would improved lysosomal functions by acNP affect palmitate lipotoxicity? Ideally these experiments should be repeated in primary cultured hepatocytes as HepG2 cells are cancerous in nature.
Response: We conducted cell viability assays in both A) HepG2 cells, and B) primary human hepatocytes. Addition of palmitate to HepG2 or primary human hepatocytes result in a 20 -25% reduction in cell viability, and treatment with acNPs (100 µg/mL) restores this cell viability ( Figure. S2F) However, in the primary human hepatocytes, higher doses of acNPs (> 120 µg/mL), results in additional cell cytotoxicity (Figure. S2G). 4. Figure S3C, the figure labeled as LAMP-1 but in the text it was stated as LAMP-2?
Response: We have changed the text to LAMP-1 to ensure consistency.
5. More experimental details should be provided for Fig S4. Response: We have included more experimental details for Fig. S4 in the revised supplementary information document, under the section "Blood and serum analysis".
6. Figure 6, the restoration of autophagy function by using western blot for LC3-II and p62 could be troublesome as both protein could be regulated at the transcription level. Also decreased LC3-II could also be due to decreased formation of autophagosomes. Adding a lysosomal inhibitor such as leupeptin in these experiments will help to clarify these issues. In addition, no data provided to show improved lysosomal functions by acNP in mouse NAFLD models.
Response: In the HepG2 cells, the addition of leupeptin does not result in a significant accumulation of LC3-II and p62 (Fig. 3E -G), but addition of bafilomycin results in a significant accumulation of LC3-II and p62. This indicates that the effect of acNPs in modulating autophagic flux is mediated mainly through changes in lysosomal acidification, and not due to modulating the lysosomal protease degradative activity. Therefore, we propose addition of a lysosomal acidification inhibition control will be more suitable to clarify the issues, as mentioned by the reviewer.
Using primary human hepatocytes, we show that under palmitate conditions (recapitulate HFD condition in mice), mitochondrial content is increased, while addition of acNPs decreases it, indicating increased mitochondrial degradation (Fig. S7K). The maximal respiratory rate (MRR) of mitochondria decrease under palmitate condition, while the addition of acNPs restores the MRR (Fig. S7L). When we measured the oxygen consumption rate (OCR) of mitochondria with and without lysosomal acidification with bafilomycin or acNPs, the OCR of mitochondria decreases upon bafilomycin treatment, while acNPs addition restores OCR (Fig. S7M). In sum, these data indicate that the effect of acNPs in restoring mitochondrial respiratory function is due to improving lysosomal acidification function, and autophagic turnover of mitochondria.
(2) Whether acNP restores in vivo lysosomal homeostasis is not shown. It is unclear whether acNP corrects metabolism through restoring liver metabolism. To address this, hepatocyte lysosomal acidification and lysosomal enzyme activities should be measured in control, LD and HD mice.
Response: Currently, there are no tools to directly measure lysosomal acidification and lysosomal enzyme activities in vivo. Therefore, we acknowledge this is a limitation in the study. To pinpoint the effect of lysosomal acidification on lysosomal homeostasis in vivo, we have treated primary human hepatocytes in the presence of bafilomycin and acNPs and measured the lysosomal activity using Magic red assay (fluorescence increases intensity as lysosomal cathepsin L activity increases) (Fig. S7N -O). Treatment with either Bafilomycin A1 or palmitate decreases Magic red intensity, indicating decreased lysosomal enzyme activity. Treatment with LD or HD acNPs increases the magic red intensity, with HD acNPs showing a more significant increase, and hence increase in lysosomal enzyme activity. These results indicate that the increase in lysosomal activity, due to treatment with acNPs, is due to changes in lysosomal acidification. 7. Inflammation is critical for the progression of NAFLD to NASH. Authors should provide data to show whether acNP can also affect liver non-parenchyma cells (such as macrophages) in addition to hepatocytes. It is highly likely a large amount of acNP would be taken up by macrophages/Kupffer cells in the liver. The function of these macrophage/Kupffer cells after acNP should be determined.
Response: We studied the co-localization of Rhodamine labelled acNPs in HFD mice with Kupffer cells stained with alexa-fluor-647-anti-mouse-clec4f-antibody (BioLegend) 14 upon tail vein injection. Through quantification of the immunofluorescence images, we observed that only 20% of the Rho-acNPs colocalized with MAC2 positive cells, while 80% of the Rho-acNPs resided in hepatocytes or the surrounding tissues. Hence, this indicates that the effect of acNPs on autophagic function as well as liver metabolic function is largely due to its action in hepatocytes. Please see the new data and results in 8. Figure 6, mitophagy data were very weak. First the control LFD group was missing, and it was unclear whether HFD would impair mitophagy in this model. The change of mitoTracker could be due to various factors such as mitochondrial membrane potential and may not be a good marker for mitophagy.
Mitophagy refereed to more specific autophagic removal of damaged mitochondria. Did the authors observed lysosomes that contain mitochondria? If HFD impaired lysosomal pH and functions, one would observe more mitochondria in the lysosomes? And acNP treatment should lead to few mitochondria inside lysosomes. The changes of more mitochondrial proteins should also be included.
Response: We thank the reviewer for the comments. We have included the LFD control, LFD with LD acNPs and LFD with HD acNPs mitochondria respirometry data (Fig. S7H -I), as well as the mitochondrial content determined using MitoTracker Deep Red (Fig. 7G -H).
We did the mitochondrial respirometry on fresh liver lysates for the HFD control, HFD and LD acNPs and HFD and HD acNPs. However, we only have frozen liver lysates for the LFD control, LFD and LD acNPs and LFD and HD acNPs. In fresh liver lysates, high dose acNPs shows an increase in MRR on pyruvate+malate (equivalent of NADH in frozen) and a slight increase in MRR on succinate. The increase in MRR that was seen in fresh lysates can be reproduced in frozen liver lysates when only maximal respiratory rate was measured. Therefore, we only measured MRR in the LFD samples. The addition of LD or HD acNPs did not change the Complex I or IV MRR in LFD mice. acNPs treatment slightly increases the maximal respiratory activity of complex II, although not statistically significant. In frozen liver lysates of HFD mice group, maximal respiratory rates of Complex I and II are less than that of LFD mice. These data show that acNPs addition does not affect mitochondrial function in LFD mice permeabilization with PFA and Triton X100. MTDR staining was preserved while MTR diffused after fixation and permeabilization. When freshly isolated mitochondria were stained with MTR or MTDR before and after depolarizing them with either FCCP or calcium overload, membrane potential disruption was only observed in MTR staining and not MTDR staining. Hence, we have shown the MTDR is a viable marker for mitochondrial protein content. The treatment of the LD or HD groups with acNPs increases the activity of both Complex I and II without affecting Complex IV. In LFD group, the mitochondrial content did not increase across all experimental groups, while the mitochondrial content of HFD group increased, and addition of acNPs decreased the mitochondrial content (Fig. 7G -H).
We have previously investigated the effect of photo-activated NPs (i.e., a nanoparticle that release  13 . We studied the effect of these nanoparticles on lysosomal reacidification on mitophagy using a mCherry-GFP-Fis1 probe, a mitochondrial chimeric protein that allows the identification of mitochondria inside autolysosomes. Under neutral pH conditions, mitochondria emit red and green fluorescence (yellow mitochondria as shown in 'BSA condition'). When mitochondria are recruited into autolysosomes, mitochondria are exposed to low pH that quenches the green fluorescent protein signal without affecting mCherry fluorescence. As shown in Fig. 3E, F from the paper below, palmitate treatment reduced the number of red mitochondria indicating a reduction of mitophagy. This effect was reversed by treatment with nanoparticles that reacidified the lysosomes. Hence, these results demonstrate the capacity of acidifying nanoparticles to restore mitophagy.